CRISPR RNA binding drives structural ordering that primes Cas7-11 for target cleavage

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Type III-E CRISPR-Cas effectors, like Cas7-11 (also known as giant Repeat-Associated Mysterious Protein), are single-subunit proteins capable of cleaving target RNAs (tgRNAs).
• These systems offer potential for precise RNA editing due to their lack of nonspecific collateral cleavage activity.

Purpose of the Study:

• To elucidate the dynamic conformational changes of apo Cas7-11 upon binding CRISPR RNA (crRNA) and tgRNA.
• To understand the mechanism of crRNA and tgRNA recognition and its impact on Cas7-11 activity.

Main Methods:

• Biochemical assays were employed to study Cas7-11.
• Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) was utilized to probe protein dynamics and conformational changes.

Main Results:

• HDX-MS revealed that crRNA binding stabilizes the folded state of Cas7-11, and subsequent tgRNA binding induces a transition to the active form.
• crRNA binding promotes the folding of insertion sequences, catalytic loops, and the Cas7.1 processing site, enhancing domain interactions.
• TgRNA binding induces conformational changes in the catalytic loops of Cas7.2 and Cas7.3, and crRNA binding facilitates TPR-CHAT binding.

Conclusions:

• Cas7-11 requires crRNA for proper folding and activation, transitioning from a stabilized state to an active conformation upon tgRNA binding.
• The interaction dynamics between Cas7-11, crRNA, and tgRNA are critical for specific RNA cleavage and editing.
• Understanding these conformational dynamics provides insights into the mechanism of Type III-E CRISPR-Cas systems for RNA editing applications.